Monoclonal antibodies (MAbs) have been generated to study the origin, differentiation, and potential function of naturally -occurring murine cytotoxic leukocytes. A rat MAb (4D11) has been generated against a novel, 87 kDa, cell surface antigen on mouse large granular lymphocytes (LGL) with natural killer (NK) activity. This antigen, LGL-1, can be detected on splenic or liver-derived LGL from most strains of mice but not on other types of lymphocytes or hematopoietic populations, and not LGL from other species. Cells electronically sorted for LGL-1 expression are highly enriched for LGL and for NK activity, whereas negatively sorted or MAb + complement-depleted LGL-1- spleen cells contained little or no NK activity. Dual-color immunofluorescence revealed subsets of LGL-1+ cells which co-expressed Ly-1 (CD5) and Thy-1, whereas CD8+ cells did not co-express LGL-1. Greater than 90% of LGL-1+ cells coexpress NK-1.1, Qa-5, and asGM1. Although LGL-1 can be found on populations of lymphokine-activated killer (LAK) cells, it has been determined that LGL-1 is not expressed on LAK precursor or effector cells. Antibody plus complement depletion of LGL-1+ cells, as well as cell sorting demonstrates that LGL-1+ cells do not contribute towards the LAK, phenomenon. LGL-1 appears to split the NK cell population into two subsets, an NK-1.1+/LGL-1+ subset that can lyse YAC-1 targets and be augmented by IL-2. The remaining NK-1.1+/LGL-1- cells, although capable of lysing YAC-1 tumor targets to a somewhat lesser degree, can respond to IL-2 and develop a broadened cytotoxic response (LAK).